Recruitment of CXCR4+ type 1 innate lymphoid cells distinguishes sarcoidosis from other skin granulomatous diseases

Sarcoidosis is a multiorgan granulomatous disease that lacks diagnostic biomarkers and targeted treatments. Using blood and skin from patients with sarcoid and non-sarcoid skin granulomas, we discovered that skin granulomas from different diseases exhibit unique immune cell recruitment and molecular signatures. Sarcoid skin granulomas were specifically enriched for type 1 innate lymphoid cells (ILC1s) and B cells and exhibited molecular programs associated with formation of mature tertiary lymphoid structures (TLSs), including increased CXCL12/CXCR4 signaling. Lung sarcoidosis granulomas also displayed similar immune cell recruitment. Thus, granuloma formation was not a generic molecular response. In addition to tissue-specific effects, patients with sarcoidosis exhibited an 8-fold increase in circulating ILC1s, which correlated with treatment status. Multiple immune cell types induced CXCL12/CXCR4 signaling in sarcoidosis, including Th1 T cells, macrophages, and ILCs. Mechanistically, CXCR4 inhibition reduced sarcoidosis-activated immune cell migration, and targeting CXCR4 or total ILCs attenuated granuloma formation in a noninfectious mouse model. Taken together, our results show that ILC1s are a tissue and circulating biomarker that distinguishes sarcoidosis from other skin granulomatous diseases. Repurposing existing CXCR4 inhibitors may offer a new targeted treatment for this devastating disease.


Figure S2 :
Figure S2: Immune cell landscape of skin granulomas.(A-B) UMAP plot of immune subcluster in unaffected and affected skin granuloma samples demonstrating tight clustering (N=55).(B) Different immune cell identities are plotted for individual conditions.(C) Dot plot of key marker genes for each cell type.Color scale represents gene expression, and dot size represents percentage of cells expressing the marker gene.(D) deCS correlation plot for cell type enrichment analysis of different cell clusters.Y-axis: main cell types identified from BlueprintEncode (left) and MonacoImmune (right) databases.Color scale represents Pearson correlation coefficient, and dot sizes represent the -log10 transformed p value.

Figure S3 :
Figure S3: Individual immune cell populations in sarcoidosis and non-sarcoidosis skin granulomas.Analysis of individual cell types with marker genes indicated below the title.Density plot demonstrates location of subgroup within UMAP.Bar plot shows relative contribution as % of total cells.DC, dendritic cell; pDC, plasmacytoid dendritic cell; cDC, classical dendritic cell; Mac, macrophage; Tc, cytotoxic T cell; Tcm, T central memory cell.Data depicted as mean ± SEM.Statistical significance was calculated using the two-tailed paired Student's t-test.*p<0.05;** p< 0.01; p*** p<0.001.

Figure S4 :
Figure S4: Sarcoidosis granulomas exhibit increased levels of ILC1s.(A) Expression levels of individual cytokines within sarcoidosis UMAP.(B) Density plot demonstrates location of B cells expressing maturation markers BCL6 and AICDA within UMAP.(C) 3D UMAP plot depicting subclustering of immune cells.ILCs are highlighted as a unique population.(D) tSNE plot depicting subclustering of immune cells.(E) Principle components analysis (PCA) of bulk RNA-seq samples of purified ILC1s from sarcoidosis skin, sarcoidosis blood, and healthy blood demonstrating separation between healthy and sarcoidosis samples.(F, G) UMAP plots of sarcoidosis affected skin from previously published datasets.(H) Bar plot shows ILC levels as % of total cells in these datasets.Statistical significance was calculated using the two-tailed unpaired Student's t-test.*p<0.05;** p< 0.01.

Figure S5 :
Figure S5: ILCs from sarcoidosis exhibit a unique activation pattern compared to ILCs from other dermatologic diseases.Comparison of gene activation pattern of ILCs across different dermatologic diseases and sarcoidosis (26).

Figure S6 :
Figure S6: Spatial transcriptomics of skin granulomas.(A) Representative H&E images of non-sarcoidosis and sarcoidosis patient skin granulomas.(B) UMAP depicting immune cell subclustering.(C) Deconvoluted cell type identification from spatial transcriptomics of sarcoidosis and non-sarcoidosis granuloma patients.Each spot is represented as a pie chart displaying relative cell proportions.Top panels highlight individual immune cell populations and bottom panels highlight ILCs specifically.(D) Expression of individual genes in sarcoidosis and non-sarcoidosis spatial transcriptomics.

Figure S8 :
Figure S8: Sarcoidosis granulomas exhibit unique signaling changes compared to nonsarcoidosis skin granulomas.(A) Global ligand-receptor analysis in non-sarcoidosis and sarcoidosis granulomas.(B-C) Cellspecific ligand-receptor analysis for non-sarcoidosis (B) and sarcoidosis (C) granulomas.Bottom half of the circular represents secreting cell types.Top half of the circle depicts receiving cell types.The inner bottom-half circle summarizes the receiving cell types by color.(D) Analysis of a specific subpopulation of fibroblasts in skin granulomas; sarcoidosis-associated populations specifically express CXCL13.(E) Differential gene expression of B cells in sarcoidosis affected skin highlights expression of CXCR5.(F) UMAP depicting expression levels of CXCR4 in sarcoidosis and non-sarcoidosis skin.Color depicts expression level.

Figure S9 :
Figure S9: Sarcoidosis patients have increased circulating ILC1s.(A) UMAP plots of PBMCs from healthy and sarcoidosis patients (N=6).(B) Cells from individual patients are annotated.(C) Bar plot showing relative contributions of different cell types in PBMCs.(D) Bar plot showing relative contribution of different cell types in individual samples.(E) Volcano plot of differential gene expression in ILCs from healthy and sarcoidosis PBMC samples.(F) NK cells levels in blood of healthy volunteers (HV), sarcoidosis patients (S) and non-sarcoidosis patients (G).Plotted as percentage of live CD45+ cells.Statistical significance was calculated using the two-tailed unpaired Student's t-test.ns, non-significant.